One important ability of proteins is to specifically bind to a specific molecule. Due to this ability, some proteins play important functions in immunoreactions and signal transmission in vivo. This ability has been the subject of intense research to develop techniques for separation and purification of useful substances using this ability. One example of such techniques already used in industrial applications is Protein A affinity separation matrices that allow for purification (capture) of high purity antibody drugs from animal cell cultures at one time.
Antibody drugs developed so far are generally monoclonal antibodies. These antibodies are mass-produced by recombinant cell-culture technology or the like. The term “monoclonal antibodies” refers to antibodies produced by clones of a single antibody-producing cell. Almost all antibody drugs currently available on the market are classified into immunoglobulin G (IgG) subclasses based on their molecular structures. Protein A is a cell wall protein produced by the gram-positive bacterium Staphylococcus aureus, and contains a signal sequence S, five immunoglobulin-binding domains (E domain, D domain, A domain, B domain, and C domain), and a cell wall-anchoring domain known as XM region (Non Patent Literature 1). In the initial purification step (capture step) in the process of antibody drug manufacture, affinity chromatography columns where Protein A is immobilized as a ligand on a water-insoluble carrier are commonly used (Non Patent Literatures 1 to 3).
Various techniques for improving the performance of Protein A columns have been developed. Various technological developments in ligands have also been made. Initially, wild-type Protein A has been used as a ligand, and currently, Protein A variants recombinantly produced by protein engineering are used as ligands in many techniques for improving the column performance. Notably, some of the Protein A engineering techniques proposed so far aim to provide Protein A ligands that can be immobilized on a water-insoluble carrier via a specific bond.
Recombinant Protein A with an additional Cys (cysteine) residue can be site-specifically immobilized on a carrier through the Cys residue (Patent Literature 1). Recombinant Protein A obtained by mutating Protein A such that the ratio between the number of Lys (lysine) residues in the antibody binding surface and the number of Lys (lysine) residues in the non-antibody binding surface of Protein A is changed can be immobilized on a carrier through multiple residues while mildly controlling the orientation of this ligand (Patent Literature 2). Recombinant Protein A with deletion of all Lys or Cys residues in the amino acid sequence can be immobilized on a carrier through its N terminal (through the α-amino group) or C terminal (through a special tag) (Patent Literatures 3 and 4). Thus, developments in technology for immobilizing a protein ligand to an affinity separation matrix are mainly based on techniques relating to Protein A columns that are required to have high performance because of their high industrial importance.